ANALYTICAL METHOD

VALIDATION

Sri Noegrohati

Good practices in
production

Principle.

Production operations must follow clearly defined

procedures in accordance with manufacturing and
marketing authorizations, with the objective of obtaining
products of the requisite quality.
All handling of materials and products, such as
receipt
cleaning
quarantine,
processing,
sampling,
packaging and labelling
storage,
distribution,
dispensing
should be done in accordance with written procedures
or instructions and, where necessary, recorded.
as far as possible no deviation from instructions or
procedures should be avoided. Any If deviations occur,
they should be done in accordance with an approved
procedure.

Monitor
Routine Commercial Manufacturing
Monitor critical operating and performance
parameters
Utilize appropriate tools, e.g., Statistical Process
Control
Monitor product characteristics (e.g., stability,
product specifications)
Monitor state of personnel training and material,
facility/equipment and SOP changes
Investigate OOS for root cause and implement
corrective action.

Validated Manufacturing Process

A validated manufacturing process has a high level of

scientific assurance that it will reliably produce
acceptable product

Measurement, Data Analysis &

Evaluation

Measurement
Appropriate systems, variables and attributes
Data Analysis
Periodic analysis to allow for trending on a periodic
basis to detect problems as early as possible and
implement corrective action
Statistical Process Control - preventative actions
Selection of Critical Quality Attributes
Meaningful specifications/ limits

The objective of an Analytical Work is:

The achievement of reliable and dependable

analytical information

Quality Assurance in Analytical

Chemistry
For conducting nonclinical laboratory studies that
support or intended to support applications for
research or marketing permits for products regulated
by the National Food and Drug Administration, Good
Laboratory Practices should be followed in a
strict sense
Compliance with this part is intended to assure the
quality and integrity of the safety data
Therefore: analytical method validation is required
Method validation provides an assurance of reliability
during normal use, and is sometime referred to as
"the process of providing documented evidence that
the method does what it is intended to do."

Analytical Method Validation

Validation the process of demonstrating that a

laboratory procedure is robust, reliable, and reproducible
in the hands of the personnel performing the test in that
Laboratory

Method validation is the process of establishing the

performance characteristics and limitations of a method
and the identification of the influences which may change
these characteristics and to what extent. It is also the
process of verifying that a method is fit for purpose, i.e.,
for use for solving a particular analytical problem.

ISO 17025:

Validation is the confirmation by examination and

the provision of objective evidence that the particular
requirements for a specific intended use are fulfilled

Why is validation important?

Validation is part of overall quality assurance program

in a laboratory to have:
the correct answer when collecting data
to have confidence that the sample contains no unsafe compound rather than there might have been
something wrong with the detection method

How does validation help with

quality assurance within a
laboratory?
Costs/Benefits of Quality Assurance

Costs
Test materials
Standards
Quality assurance
equipment
Analysis of QA/QC
samples
Quality assurance
official
Committee Work
Interlab Studies
Travel to meetings

Benefits

More efficient outputs

Fewer replicates for
same
reliability
Fewer do-overs
Greater confidence of:
Staf
Laboratory
Customers

Why is Method Validation

Necessary?

It is an important element of quality control.

Validation helps provide assurance that a measurement
will be reliable.
In some fields, validation of methods is a regulatory
requirement.
The validation of methods is good science.

When is Validation needed?

Before introduction of a new method into routine use

Whenever the conditions change for which a method
has been validated, e.g., instrument with diferent
characteristics
Whenever the method is changed, and the change is
outside the original scope of the method

Who carries out method

validation?
Can laboratories validate methods on their own, in-house
validation
The in-house methods should demonstrate that
a. the required tolerances of all measurements undertaken
within the method (volume, temperatures, masses etc.);
b. the forms of the determinand measured, including
speciation;
c. the efect of interferences has been widely investigated
and quantified;
d. significant sources of error have been identified and
adequate means of controlling them have been identified.
questions
Will methods validated in this way be recognized by other
laboratories?
What sort of recognition can be expected for in-house
methods used in a regulatory environment?

Error in Chemical Analysis

Real Population

Accurate
Precise
Error: Systematic
Random

Estimate of
Sample

XS

Reliable method the

obtained results are
accurate and correctly
reflect the sample being

Gross error
Errors which are so serious
that there is no real
alternative to abandoning
the experiment and
making a complete start
Random error
Error that cause the
individual result to fall on
both side of the average
value affect the
precision of an experiment
Systematic error
Error that cause all the
result to be in error in the
same sense affect the
accuracy, i.e. proximity to
the true value

Choosing, developing and evaluating methods

Analytical Method

Steps to have an Analytical Information:

Sampling and sample storage
Preparation of Samples
Measurement
Equipment and Equipment parameters
Reagents
System Suitability testing
Analytical
Method
Preparation of Standards
Analytical
Procedure
Procedure
Evaluation
Analytical Principle
Calculations
Reporting of results
Documentation

QUALITY ASSURANCE

Assure that:

What is reported there is really there

The level is exactly as what you report
There is no addition through contamination
If reported as negative, it is really not there

Method Selection

Appropriate for intended use

Preferable standard method
Published in international, regional national

standard, technical journals

Non Standard methods required

validation
Journal article
In-house method

Validation Parameters

For a large number of replicates the results

approach GAUSSIAN or NORMAL
DISTRIBUTION CURVE
The mean value
x
gives the center of
the distribution
The standard
deviation s measures
the width of the
distribution

Experimental results are

commonly expressed in
the form:
mean standard
deviation
_

Sri Noegrohati,
x s Fac
of Pharmacy

17

GMU

Normal Distribution

20

40

60

80

100

exp x / 2 2
y
2

Sri Noegrohati, UGM

Curve is symmetrical and

centred at m.
The greater the value of
, the greater the spread
of the curve.
Whatever values of and
,
68.27% of observations
are within
95.45% of observations
are within 2
99.97% of observations
are within 3
18

Definitions

Arithmetic mean: average of all observations

If the sample is random then the
n
arithmetic mean is the best
xi
estimate of the population (true)
x i 1
mean, m
n

Variance: measures the extent to which the data

difers in relation to itself. Variance of population is the
mean squared deviation from the population mean,
denoted 2, while the variance of the sample data is
denoted s2. n
n

2
Sri Noegrohati, UGM

x
i
i 1

x
i
i 1

n 1
19

Accuracy

The closeness of agreement between a test

result and the accepted reference value.
Accuracy of a measuring instrument is the
ability of a measuring instrument to give
responses close to a true value.
EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics, pp. 39, 41;
available at http://www.eurachem.ul.pt/guides/valid.pdf

SYSTEMATIC ERROR
Reproducible under the same conditions in the
same
experiment
Can be detected and corrected for
It is always positive or always negative

To detect a systematic error:

Use Standard Reference Materials

Run a blank sample
Use diferent analytical methods
Participate in round robin experiments
(diferent labs and people running the same
analysis)
21

Sri Noegrohati,
Fac of Pharmacy
GMU

Precision
The closeness of agreement between independent
test results obtained under stipulated
conditions.
Precision depends only on the distribution of
random errors and does not relate to the true value
or specified value. The measure of precision is usually
expressed in terms of imprecision and computed as a
standard deviation of the test results.
A measure for the reproducibility of
measurements within a set, that is, of the scatter or
dispersion of a set about its central value.

EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods:

A Laboratory Guide to Method Validation and Related Topics, p. 45;
available at ttp://www.eurachem.ul.pt/guides/valid.pdf

RANDOM ERROR
Uncontrolled variables in the
measurement
Can be positive or negative
Cannot be corrected for
Random errors are independent of each
other

Random errors can be reduced

by:

Better experiments (equipment, methodology,

training of analyst)
Large number of replicate samples

23

Sri Noegrohati,
Fac of Pharmacy
GMU

Student's t-Tests

"Student's t-test deals with the problems associated with

inference based on "small" samples: the calculated mean
(Xavg) and standard deviation () may by chance deviate
from the "real" mean and standard deviation (i.e., what
you'd measure if you had many more data items: a
"large" sample).
For example, it it likely that the true mean size of maple
leaves is "close" to the mean calculated from a sample of
N randomly collected leaves.
At 95% of the time, the actual mean would be in the
range:
If N=5, Xavg 2.776 /N1/2;
if N=10: Xavg 2.262 /N1/2 ;
if N=20: Xavg 2.093 /N1/2 ;
if N=40; Xavg 2.023 /N1/2 ;
and for "large" N: Xavg 1.960 /N1/2 .
(These "small-sample" corrections are included in the
descriptive statics report of the 95% confidence interval.)
Student" (real name: W. S. Gossett [1876-1937])

CONFIDENCE INTERVALS
The confidence interval is the expression stating
that the true mean, , is likely to lie within a
certain distance from the measured mean, x.

Students t test

The confidence interval is given

by:
_
ts

Where t is the value of students t taken from the

table

26

Sri Noegrohati,
UGM

Students t table
d.f.

90%

95%

99%

99.9%

2
3
4
5
10
20
30

2.920
2.353
2.132
2.015
1.812
1.725
1.697

4.303
3.182
2.776
2.571
2.228
2.086
2.042

9.925
5.841
4.604
4.032
3.169
2.845
2.750

31.596
12.941
8.610
6.869
4.587
3.850
3.646

Worked example:
Fluoride content of a sample determined potentiometrically in
water is (mg/l) 4.50, 3.80, 3.90, 4.20, 5.00 and 4.80 for
separate analyses.
Mean = 4.37
Standard deviation = 0.48
90% confidence limits are: = 4.37 2.015 x (0.48/6) = 4.37
0.39
27
Sri
Noegrohati,
UGM
99%
confidence
limits are: = 4.37 4.032 x (0.48/6)
= 4.37

Sri
Noe
gro
hati
,
Fac
of
Pha
rma
cy
GM
U

A t test is used to compare sets of

measurements.
Usually 95% probability
is good enough.
28

APPLYING STUDENTS
T:
1) COMPARISON OF MEANS
Comparison of a measured result with a
known (standard) value

t calc

known value x

tcalc > ttable at 95% confidence level

results are considered to be different
the difference is significant!

Statistical tests are giving only probabilities.

ey do not relieve us of the responsibility of interpreti
our results!
29

2) COMPARISON OF REPLICATE
MEASUREMENTS
For 2 sets of data
with number of
measurements n1 , n2 and means x1, x2 :

t calc

x1 x 2
n1n2

spooled n1 n2

Where Spooled = pooled std dev. from both sets of data

spooled

s12 (n1 1) s22 (n2 1)

n1 n2 2

tcalc > ttable at 95% confidence level

difference between results is significant.
Degrees of freedom
= (n1 + n2 2)
30

Sri Noegrohati,
Fac of Pharmacy
GMU

A Comment on Minimum Numbers

of
Samples
Impact of Number of Experiments on Capturing Variability in a
Population of Data

Number of Samples Needed

Relationship between a sample and a population
of data

Q TEST FOR BAD DATA

Q calc

gap

range

The range is the total

spread of the data.
The gap is the difference
between the bad point
and the nearest value.
Example:
12.2

Gap

12.4 Range
12.5 12.6
12.9

If Qcalc > Qtable

discarded questionable

33

Sri
Noe
gro
hati
,
Fac
of
Pha
rma
cy
GM
U

The VAM Principles

VAM = Valid Analytical Measurement
1. Analytical measurements should be made to satisfy an

agreed requirement.
2. Analytical measurements should be made using
methods and equipment that have been tested to
ensure they are fit for their purpose.
3. Staff making analytical measurements should be
both qualified and competent to undertake the
task.
4. There should be a regular and independent assessment
of the technical performance of a laboratory.
5. Analytical measurements made in one location should
be consistent with those made elsewhere.
6. Organizations making analytical measurements should
have well defined quality control and quality assurance
procedures.

The Analytical Approach

Statement of Problem
Definition of Objective
Selection of Procedure
Sampling, Sample Transport and Storage
Sample Preparation
Measurement/Determination
Data Evaluation
Conclusions and Report

35

Sri Noegrohati,
UGM

Assumptions When Performing Validation

The equipment on which the work is being done is

broadly suited to the application. It is clean, well
maintained and within calibration.
The staf carrying out the validation are competent in
the type of work involved.
There are no unusual fluctuations in laboratory
conditions and there is no work being carried out in
the immediate vicinity that is likely to cause
interferences.
The samples being used in the validation study are
known to be sufficiently stable.

System Suitability

Fitness for purpose is the degree to which data

produced by a measurement process enables a
user to make technically and administratively
correct decisions for a stated purpose.

Calibration of the instrument

Based on Lambert - Beer law linear correlation between

absorbance and concentration calibration curve

Finding the Best

Straight Line:
a line that
minimises the
deviations in the
y-direction using
the sum of the
square of these
deviations)method of least
squares.
Sri Noegrohati, UGM

39

Linearity and Range

Linearity defines the ability of the method to obtain

test results proportional to the concentration of
analyte.
The Linear Range is by inference the range of
analyte concentrations over which the method gives
test results proportional to the concentration of the
analyte.
Working range is a set of values of measurands for
which the error of a measuring instrument is intended
to lie within specified limits.

METHOD OF LEAST SQUARES

Assumption:
There is a linear relationship
Errors in the y-values (measured values) are greater than the
errors in the x-values minimise only the vertical deviations.
Uncertainties for all y-values are the same

minimise only the vertical deviations

Equation of a straight line: y = mx + c
where m = slope and c = y-intercept

x y x y x
c
n x x

n xi yi xi yi

n xi2 xi 2

41

i
2

i i
2
i

Sri Noegrohati,
UGM

Estimation of the std. dev. for all y

values

The vertical deviation can be calculated as follows:

di = yi (mxi + c)

Some deviations are positive (point lies above the

curve) and some are negative (point lies below the
curve) square the values so that the sign does not
play a role

di2 = (yi mxi - c)2

Estimate the standard deviation for all y values

sy
Sri Noegrohati, UGM

d
i

n2

42

Standard deviation for the slope

(m):
2

sm

sy n

n xi2 xi 2

Standard deviation for the intercept (c):

2

sc

s y2 xi2

n xi

Detectability
Is the Analytical Signal distinguishable from the Blank?
need to know the uncertainty of the
measurements.sc

Y detection limit = c+3sc LOD = (c+3sc)-c/m

43

Sri Noegrohati,
UGM

Sensitivity:
calibration sensitivity = slope (m) of calibration curve.=
ability to discriminate between small diferences in analyte
concentration
analytical sensitivity (g) = slope (m)/standard deviation
(Ss)

lnear
range,
where the response
of the analyte is
linear
with
concentration.
The dynamic
range is the
concentration
where there is a
measureable
response to the
analyte, even if it is
Sri Noegrohati,
not linear.
44

UGM

Linear Dynamic Range: linear region of calibration curve

where the lower limit is ten times the standard deviation of
the blank.
LOQ - limit of quantitation
LOL - limit of linearity

Concentration (mM)
45

Sri Noegrohati,
UGM

Sensitivity
Calibration of the fundamental Analytical Procedure

Limit of detection (LOD) the concentraton that can

be measured with reasonable statistical certainty of
significantly diferent from blanc signal.
Limit of quantitative measurement (LOQ) the
lowest concentration of an analyte that can be
determined with acceptable precision (repeatability)
and accuracy under the stated conditions of the test
using the chosen analytical procedure.

Limit of Linear Response (LOL)

Point of saturation for an instrument detector, so that higher

amounts of analyte do not produce a linear response in signal

How is this
best done
in
practise?

Reproducibility

Reproducible method the same or very similar

results are obtained each time a sample is tested
Precision under reproducibility conditions, i.e.
conditions where test results are obtained with the
same method on identical test items in diferent
laboratories with diferent operators using diferent
equipment.
Will you get the same result each time you test a
sample?
Diferent from repeatability, which is the precision
under repeatability conditions, i.e. conditions where
independent test results are obtained with the same
method on identical test items in the same laboratory
by the same operator using the same equipment
within short intervals of time.

Robustness (Ruggedness)

Robust method successful results are obtained a

high percentage of the time and few, if any, samples
need to be repeated
The robustness of an analytical procedure is a
measure of its capacity to remain unafected by
small, but deliberate variations in method parameters
and provides an indication of its reliability during
normal usage.
You do not want the method to fail when you only
have enough material for a single try.

Specificity

The ability of a method to measure only what it

is intended to measure.
Specificity is the ability to assess unequivocally
the analyte in the presence of components which
may be expected to be present. Typically these
might include impurities, degradants, matrix,
etc.

Stability

Will the method produce a result reliably over

time?
Control chart i.e. Shewhart chart, are an efective
tool for monitoring stability and quality
assurance over time

Development of Analytical
Method
Funded by the
Company

Performed
by
manufacture
r

Performed by
Lab

Common Perceptions of
Validation